Comparison of IL-10 gene promoter polymorphisms and haplotypes between high-grade squamous intraepithelial lesions or cervical cancer and negative cervical cytology

Abstract

Cervical cancer, a leading cancer among women, is strongly associated with Human Papillomavirus infection, but host genetic factors also contribute to the progression from high-grade squamous intraepithelial lesions (HSIL) to invasive cancer. Interleukin-10 (IL-10), an immunosuppressive cytokine, may influence susceptibility to HSIL and cervical cancer through genetic variations. This study aimed to compare IL-10 gene promoter polymorphisms, -1082 A > G and − 819T > C, in women diagnosed with HSIL or cervical cancer and those with negative for intraepithelial lesion or malignancy (NILM). In this case-control study, 309 women were analyzed, including 142 with HSIL or cervical cancer and 167 controls with NILM. Blood samples were collected for DNA extraction and genotyping of polymorphisms through PCR amplification. Statistical analyses included comparisons of genotype and allele frequencies, haplotype frequency, and assessments of Hardy-Weinberg equilibrium and linkage disequilibrium. The mean age was 33.4 years for cases and 41.7 years for controls (p < 0.05). For the − 1082 A > G polymorphism, the GG genotype was significantly associated with a decreased risk of HSIL and cervical cancer (p = 0.0266, OR = 0.35). Recessive model (GG vs. AA + AG) confirmed this association (p = 0.0045, OR = 0.29). AC/GC diplotype was associated with a 2-fold increased risk of cervical lesions. Further studies are needed to confirm our results.

https://orcid.org/0000-0002-1344-0078.

Introduction

Globally, cervical cancer is the second most prevalent cancer among women, following breast cancer¹. Human Papillomavirus (HPV) infection is a necessary factor in the development of high-grade squamous intraepithelial lesions (HSIL) and their subsequent progression to cervical cancer, but it is insufficient on its own to cause malignancy. Additional contributory factors are required for this progression². Evidence suggests that a limited immune response to HPV associated with host genetic factors may contribute to the carcinogenesis, increasing the risk of HSIL progression to invasive carcinoma^3,4.

Several immunological components, including immunosuppressive cytokines such as interleukin (IL)-10, play a significant role in modulating the immune response to HPV. These factors affect HPV acquisition, viral persistence, and the regression or progression of HSIL within the cervical microenvironment⁵. High-risk HPV genotypes alter immune system cells to promote an immunosuppressive microenvironment, leading these cells to express IL-10⁶. Furthermore, IL-10 downregulates pro-inflammatory cytokine responses, including tumor necrosis factor-alpha (TNFα), IL-1, IL-6, IL-8, IL-12, and interferon-gamma (IFN-γ), thereby mitigating tumor growth^8,9. IL-10 itself inhibits the expression of IFN-γ from Th1 cells, increasing antigen presenting cells inactivation¹⁰a process associated with the progression of carcinogenesis. Studies have demonstrated that IL-10 promotes tumor invasiveness and regulates angiogenesis in malignant cells^11,12. Curiously, an increased level of IL-10 expression has been reported in women with HSIL^13,14.

Studies hypothesized that single nucleotide polymorphisms (SNPs) in genes associated with immune response may be involved in cervical cancer etiology^15–18. The contribution of IL-10 to carcinogenesis has focused the effects of SNPs in the promoter region. Serum levels of IL-10 are influenced by at least three polymorphic sites within its promoter. Gene encoding for IL-10 is located on human chromosome 1 (1q31 and 1q32), it spans 4.8 kb, consisting of 5 exons and 4 introns, that encode a total of 178 amino acids¹⁹. Over 50 polymorphic loci have been documented for this gene. The three most common SNPs in IL-10 gene promoter region are − 1082 A > G (rs1800896), -819T > C (rs1800871) and − 592 C > A (rs1800872), which have been identified as significant regulators of transcription levels of IL-10 messenger RNA and the IL-10 expression^18,20. These SNPs have been associated with an elevated risk of developing cervical cancer¹⁷. However, the relationship between these polymorphisms and susceptibility to HSIL and cervical cancer remains contentious, as research findings have produced inconsistent results^21,22.

Several reports have been demonstrated by in vitro and in vivo analysis that the change from allele A to G promotes upregulation of IL-10 transcription. Specifically, the homozygous GG genotype is associated with increased IL-10 expression²³. On the other hand, AA genotype downregulates, and heterozygous AG is associated with a medium cytokine expression²⁴. Currently, studies have demonstrated that ethnicity and cytokine polymorphism are key factors in susceptibility to HSIL and cervical cancer²⁵. Therefore, in this study, we aimed to perform a genetic analysis of -1082 A>G and − 819T>C SNPs affecting IL-10 promoter genes regions in peripheral blood samples obtained from Brazilian females to compare these polymorphisms in women diagnosed with HSIL or cervical cancer with controls negative for intraepithelial lesion or malignancy (NILM).

Results

Patients’ characteristics

A flowchart of the recruitment process is presented in Fig. 1. Among the 309 participants in this study, 142 patients with HSIL and cervical cancer were compared with 167 women with NILM. The mean age was 33.4 ± 10.3 years in case group, and 41.7 ± 10.8 years in control group (p < 0.05). Moreover, most women in both the case and control groups were black (61.1% and 64.4%, respectively).

Fig. 1.

Study selection Strobe flowchart.

Polymorphism of the − 1082 and − 819 promoter region of the IL-10 gene

The frequencies of alleles and genotypes of the − 1082G/A and − 819 C/T polymorphisms of IL-10 between patients with histologically confirmed HSIL or cervical cancer and those with NILM are summarized in Table 1. Regarding SNP − 1082G/A, we observed significantly different distributions only for the genotype frequencies between the case and control groups, as the allele frequencies were similar. Therefore, an association was detected for the GG genotype (p = 0.0266, OR = 0.35), indicating differential distribution of this SNP in patients with cervical lesions. The power of association for the GG genotype was greater when the recessive model was included in the analysis ((AA + AG) × GG; p = 0.0045, OR = 0.29), indicating a protective effect in the case group compared with the control group. On the other hand, for the − 819 C/T SNP, no significant differences were found between the groups evaluated for both genotypes and alleles.

Table 1.

Distribution of alleles and genotypes of SNPs in the IL-10 gene promoter. N, number of individuals. OR, odds ratio; CI, confidence interval. Statistics used was fisher’s exact two-tailed test. Bold values denote differences statistically significant with P < 0.05. A significant difference was also found in AG vs. XL (p = 0.015, 0R = 4.0 (1.6–9.9).

Polymorphism	Case		Control
	N = 142	(%)	N = 167	(%)	p-value	OR (95% CI)
Alleles (-1082 A/G)
A	194	68.3	217	65.0	1.0 (Ref)
G	90	31.7	117	35.0	0.3935
Genotypes
AA	59	41.5	75	44.9	1.0 (Ref)
AG	76	53.5	67	40.1	0.1493
GG	7	4.9	25	15.0	0.0266	0.35 (0.14–0.88)
Dominant model
AA	59	41.5	75	44.9	1.0 (Ref)
(GG + AG)	83	58.5	92	55.1	0.5667
Recessive model
(AA + AG)	135	95.1	142	85.0	1.0 (Ref)
GG	7	4.9	25	15.0	0.0045	0.29 (0.12–0.70)
Alleles ( -819 C/T)
C	183	64.4	209	62.6	1.0 (Ref)
T	101	35.6	125	37.4	0.6754
Genotypes
CC	58	40.8	71	42.5	1.0 (Ref)
CT	67	47.2	67	40.1	0.4593
TT	17	12.0	29	17.4	0.3885
Dominant model
CC	58	40.8	71	42.5	1.0 (Ref)
(TT + CT)	84	59.2	96	57.5	0.8172
Recessive model
(CC + CT)	125	88.0	138	82.6	1.0 (Ref)
TT	17	12.0	29	17.4	0.2024

Regarding the Hardy-Weinberg equilibrium (HWE) for the − 819 C/T SNP, no significant differences were found in the genotype frequencies between the two groups. However, the genotypic distribution of the − 1082G/A SNP did not conform to HWE only in the case group, where the G allele in the homozygous form appears to be becoming rare due to the influence of evolutionary factors (X² = 7.92) (Table 2).

Table 2.

Distribution of alleles and genotypes of SNPs in the IL-10 gene promoter.

	Case			Control
Genotypes (-1082 A/G)	Expected	Observed	X 2	Expected	Observed	X 2
Common homozygotes (AA)	66.26	59	7.92	70.49	75	2.35
Heterozygotes (AG)	61.48	76		76.01	67
Rare homozygotes (GG)	14.26	7		20.49	25
Genotypes (-819 C/T )
Common homozygotes (CC)	58.96	58	0.12	65.39	71	3.44
Heterozygotes (CT)	65.08	67		78.22	67
Rare homozygotes (TT)	17.96	17		23.39	29

Linkage disequilibrium

Figure 2 illustrates the linkage disequilibrium (LD) map of the two SNPs (-1082G/A and − 819 C/T). A deviation coefficient (D’) value of 0.96 was observed, indicating a strong LD among the evaluated SNPs.

Fig. 2.

Haploview representation of LD as a function of D’ among the SNPs of the IL-10 gene.

Haplotypes and diplotypes of the − 1082 and − 819 polymorphism of the IL-10 gene

Analysis of two SNPs in IL-10 revealed the existence of four haplotypes. A lower haplotype frequency (-1082G and − 819T) was observed in both the case and control groups. Furthermore, no significant association was found between the two groups in terms of haplotype distribution (Table 3). When the combined haplotypes (diplotypes) were analyzed, eight possibilities were identified. The AC/GC diplotype was associated with a 2.17-fold increased risk of cervical lesions compared with the other diplotypes (p = 0.0099). Conversely, the GC/GC diplotype was less frequent (4.9% vs. 14.4%; P = 0.0072, OR = 0.30) in women with cervical lesions than in the control group (Table 3) (Fig. 3).

Table 3.

Frequency of haplotypes and diplotypes of SNPs in the IL-10 gene promoter. N, number of individuals; OR, odds ratio; CI, confidence interval. Statistics used was fisher’s exact two-tailed test; bold values denote differences statistically significant with p < 0.05.

	Case		Control		p-value	OR (95% CI)
	N = 142	(%)	N = 167	(%)
Haplotypes ( -1082/-819 )
AC	93	32.7	94	28.1	0.2200
AT	101	35.6	123	36.8	0.8012
GC	90	31.7	115	34.4	0.4935
GT	0	0.0	2	0.6	-
Diplotypes ( -1082 / -819 )
AC/AC	13	9.2	23	13.8	0.2190
AC/AT	29	20.4	24	14.4	0.1749
AC/GC	38	26.8	24	14.4	0.0099	2.17 (1.23–3.85)
AT/AT	17	12.0	28	16.8	0.2600
AT/GC	38	26.8	42	25.1	0.7949
AT/GT	0	0.0	1	0.6	-
GC/GC	7	4.9	24	14.4	0.0072	0.30 (0.13–0.74)
GC/GT	0	0.0	1	0.6	-

Fig. 3.

Genotype distribution for (A) rs1800896 and (B) rs1800871 across global populations.

Bioinformatic analysis

The analysis demonstrated that, across diverse global populations, the frequency of the rs1800896 GG genotype, associated with a reduced disease risk in our Brazilian cohort, was consistently lower among cases and significantly higher among controls. This pattern was particularly pronounced in African, European, and Middle Eastern populations, thereby reinforcing a protective association that aligns with a recessive model. Comparable, albeit less pronounced, trends were observed for rs1800871. The genotype frequency bar plots provided a clearer visual distinction between case and control groups compared to raw count plots, particularly in admixed populations. These findings not only substantiate the protective role of the rs1800896 GG genotype within the Brazilian population but also suggest that this effect may extend to other ancestries, thereby enhancing the potential relevance of this association.

Discussion

The finding that some individuals are genetically predisposed to high IL-10 expression suggests their involvement in carcinogenic process²⁴. Several genetic polymorphisms have been associated with susceptibility to cervical cancer. Our analyses focused on whether IL-10-1082G/A and − 819 C/T polymorphisms are associated with HSIL and cervical cancer, and we found a significant association between the − 1082G/A variant. However, the − 819 C/T SNP was not significantly associated with HSIL and cervical cancer.

Alterations in cytokine-related coding genes may present a carcinogenic mechanism by altering their expression^26,27. Studies have shown that polymorphisms in the IL-10 gene influence the expression of this cytokine, leading to both protective and promoting effects²⁸. An increasing body of evidence suggests that IL-10 exerts antitumor effects by inhibiting angiogenesis and tumor growth^29,30. Our present analysis demonstrated that the − 1082G/A allele in homozygosity was associated with increased IL-10 expression, with a significant difference between the groups. We also found an increased association in the recessive model, considering the combination (AA + AG) vs. GG, suggesting that the GG genotype contributes to a lower risk of progression for cervical lesions than the AA or AG genotypes. Rarefaction of the G allele in homozygosity in the case group for the − 1082G/A polymorphism may indicate selective pressure or the influence of evolutionary factors that modify the frequency of this allele in individuals with cervical lesions.

This phenomenon can be elucidated through a threshold mechanism, which posits that only individuals possessing two copies of the G allele (GG) achieve adequate levels of IL-10 expression to mount a protective immune response. To assess whether this association is applicable beyond our specific cohort, we conducted an analysis of genotype distributions across a variety of populations. Our findings indicated that the frequency of the GG genotype at rs1800896, the same variant associated with protection in the Brazilian cohort, was consistently lower among cases and higher among controls, particularly within African, European, and Middle Eastern populations. These patterns substantiate the protective role of the GG genotype under a recessive model and imply that this effect may extend to other ancestral backgrounds. These results underscore the broader significance of our association and suggest potential evolutionary or population-level mechanisms that may influence susceptibility to disease.

Our findings provide strong evidence that the GG genotype may have a protective effect against the development of cervical lesions. Convincing evidence has indicated elevated levels of IL-10 in the G allele. We suggest that the antitumor role implicated in NK cell activation, T cells, macrophages, and nitric oxide³¹ may reduce the risk of HSIL progression to invasive cervical cancer.

Although several studies reported no association between the − 1082G/A polymorphism and an increased risk of cervical cancer susceptibility^32–34Guo et al.²⁰ performed a meta-analysis of 17 studies involving a total of 7.286 women and found a significant association between the − 1082G/A polymorphism and an increased risk of cervical cancer development, especially in Asians. The researchers concluded that ethnic diversity may be a potential factor for heterogeneity. Thus, considering that the frequency of gene polymorphisms varies in different racial/ethnic groups, the effect of the polymorphism at position − 1082 A > G in the promoter region of the IL-10 gene on HSIL and cervical cancer regression or protection has been described in different populations, such as Indian, Zimbabwean, Hungarian, and Japanese. On the other hand, studies in Dutch, Argentine, South African, Chinese, South Korean, and Tunisian populations found no significant association^{14,25,35–39}.

In the Indian and Zimbabwean populations, the genotypes at position − 1082 A > G corresponding to high and intermediate expression, GG and AG, respectively, were significantly associated with the severity of cervical cancer^35,36. In the Hungarian population, the GG genotype may decrease susceptibility to cervical abnormalities unrelated to HPV infection³⁷. Matsumoto et al.⁴⁰ indicated that the carrier frequency of IL-10 − 1082 G alleles (genotypes GA or GG), which are associated with higher IL-10 expression, significantly increased with the severity of cervical cancer in Japanese women.

We also reported that the − 819 C/T polymorphism showed no significant differences in genotype and allele frequencies between the case and control groups, suggesting that − 819 C/T may not be associated with the risk of HSIL and cervical cancer. A recent study involving women from northeastern Brazil found no significant differences in genotypic and allelic frequencies at the − 1082G/A and − 819 C/T loci between HPV-infected patients (with HPV types 18, 31, and 58) and HSIL, LSIL, and healthy controls⁴¹. Additionally, our results differ from those of Bai et al.⁴²who showed an association between the − 819TT SNP and the risk of cervical cancer in the Chinese population but did not find this association for the − 1082G/A polymorphic site.

Moreover, the influence of individual genetic variants on carcinoma development is hypothesized to be weaker than that of multiple allele arrangements. Few studies have examined the association between IL-10 gene polymorphisms and susceptibility to cervical cancer. Our findings showed that both analyzed polymorphisms were strongly in linkage disequilibrium, suggesting that these variants are genetically associated and tend to be inherited together with susceptibility to HSIL and cervical cancer. Although recombination frequency is not strictly proportional to chromosomal distance and is sensitive to ancestral effects, our findings are not surprising given that they are only 263 nucleotides apart and there are no intervening introns.

Consequently, in the present study, we performed haplotype analysis for the − 1082G/A and − 819 C/T polymorphic sites and observed that the overall haplotype frequency was low and did not show a significant association. The fact that it occurs less frequently suggests a lower likelihood of being associated with the risk of cervical lesions. Another study involving Brazilian women reported that the AC haplotype was significantly more frequent in HPV-infected patients than in control group³⁴. Nevertheless, our analysis showed a significant association between the AC/GC diplotype and an increased risk, and the reduced frequency of the GC/GC diplotype in the case group indicates that these combinations have a different impact on the risk of cervical lesions. The AC/GC diplotype may be associated with a 2.17-fold increased risk of cervical lesions owing to possible combined genetic effects or functional interactions between the variants. In contrast, the GC/GC diplotype may have a protective effect or may not be associated with risk.

To our knowledge, this is the first case-control study to evaluate IL-10 -1082G/A and − 819 C/T genotypes and haplotypes in Brazilian women with HSIL or cervical cancer compared to those with NILM cytology. Despite its relevance, the study has some limitations that warrant consideration. The absence of HPV DNA testing in both groups limits the assessment of viral status as a confounding factor. The modest sample size may have reduced statistical power and affected the detection of subtle associations. Additionally, cross-sectional design does not permit causal inferences. Grouping HSIL and cervical cancer cases may have introduced selection bias and limited the generalizability of the findings. Moreover, the lack of functional assays prevents confirmation of the biological role of the IL-10 -1082GG genotype. Further studies incorporating functional analyses—such as promoter activity, cytokine quantification, and genotype-expression profiling—are needed to elucidate the underlying mechanisms.

Conclusions

The comparison between the groups suggests a possible association between the AC/GC diplotype and a twofold increased risk of developing HSIL and cervical carcinoma. Conversely, the GC/GC diplotype may have a protective effect or may not be associated with risk. To confirm these possibilities, prospective studies involving healthy women and different types of cervical lesions, separated into groups, are needed. With adequate follow-up and rigorous bias control, it may be possible to determine whether IL-10 promoter gene polymorphisms increase or decrease the risk of cervical cancer.

Methods

Study design and population

This case-control study involved 309 adult women who underwent cervical cancer screening at two hospitals in Northeast Brazil. This study included 142 women histologically diagnosed with HSIL and cervical cancer. Simultaneously, a control group of 167 women with cervical cytology NILM was recruited. Inclusion criteria included women aged over 18 years, non-smokers, and those with no history of cancer, chronic illness, or immune disorders. Participants who had others gynecological tumors, pregnant or had undergone a prior hysterectomy were excluded. Informed written consent was obtained from all participants for the use of peripheral blood samples for genotyping in this study. The study protocol was approved by the institutional ethics committee (protocol number 68615417.0.0000.5293) and all methods were performed in accordance with the relevant guidelines and regulations.

DNA extraction

Venous blood samples were used for genomic DNA extraction following the Salting-out protocol⁴³. The samples were stored at -20 °C until use. The extracted DNA was diluted to a final concentration of 100 ng/µL after quantification, and purity was determined using a Nanodrop^® spectrophotometer.

Genotyping of IL-10–1082 A/G and − 819 C/T polymorphisms

Polymorphic sites in the promoter region − 1082G/A (rs1800896) and − 819 C/T (rs1800871) were analyzed by conventional polymerase chain reaction (PCR) amplification in a final volume of 25 µL containing 1× reaction buffer (20 mM Tris-HCl, 50 mM KCl, 3.0 mM MgCl2), 0.2 mM of each dNTP, 0.4 pmol of each primer (IL-10 up and IL-10 down), 2.0 U of recombinant Taq DNA Polymerase (Invitrogen, California, USA), 1.6% DMSO, and 100 ng of genomic DNA. PCR was carried out in a SimpliAmp™ thermocycler (Applied Biosystems) using the following program: initial denaturation at 94 °C for 3 min, followed by 40 cycles at 94 °C for 45 s, 54 °C for 45 s, 72 °C for 1 min, and a final elongation step at 72 °C for 7 min.

Amplified products were monitored by 2% agarose gel electrophoresis and SYBR^® Green staining (Life Technologies, USA). PCR products containing fragments of approximately 650 bp were directly sequenced with the IL-10 down primer and added to the DMSO reaction on an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster City, California), using the BigDye terminator version 3.1 cycle sequencing kit (Applied Biosystems). The two polymorphisms identified in IL-10 gene were counted individually.

Statistical analysis

The sample size of 290 participants was chosen to ensure a maximum error of ± 3% points with 95% confidence. We compared the distributions of genotypes and alleles for all SNPs between the case and control groups. Allele and genotype frequencies were determined by direct counting. Haplotype frequencies in both groups were estimated using the PHASE (version 2.1) and ARLEQUIN (version 3.5; expectation maximization algorithm, EM) programs. Haplotype inferences with probabilities greater than 0.98 were considered for further analysis.

We examined the differences between the groups using either a Chi-square or Fisher-exact test for categorical variables, or an independent sample t-test for continuous variables. A p-value < 0.05 was considered statistically significant, and the odds ratio (OR) with a 95% confidence interval (95% CI) was calculated. Contingency table analyses were performed using GraphPad Prism version 5.01 for Windows (GraphPad Software, San Diego, CA, www.graphpad.com). HWE was assessed for each SNP to determine whether the observed genotype frequencies in the control group deviated from those expected under HWE⁴⁴. To construct the LD map, we used Haploview (version 4.2). The degree of genetic linkage between two SNPs was estimated using Lewontin’s standardized deviation coefficient (D’) > 0.9.

We performed a comprehensive bioinformatic analysis using globally available datasets. Curated genotype and allele frequency data for the IL-10 promoter polymorphisms rs1800896 and rs1800871 were obtained from Guo et al.²⁰, the Human Genome Diversity Project (HGDP)⁴⁵, the 1000 Genomes Project (1KGP)⁴⁶, and ClinVar annotations⁴⁷. Only genotypes with clear phenotypic classification into high-grade squamous intraepithelial lesions (HSIL; cases) or negative for intraepithelial lesion or malignancy (NILM; controls) were included. Data were extracted and filtered by ethnic background, and populations with insufficient sample sizes (e.g., Oceanic and Native American) were excluded.

For each SNP and population, we calculated genotype counts and allele frequencies, and applied Fisher’s exact test to evaluate differences in genotype distributions between case and control groups⁴⁸. Analyses were conducted under a recessive model (i.e., comparing GG vs. AA + AG for rs1800896) using a 2 × 2 contingency table. Multiple testing correction was applied using the Benjamini-Hochberg false discovery rate procedure⁴⁹, and adjusted p-values were annotated using the conventional three-star significance notation. All analyses were conducted in Python, with visualizations finalized in Inkscape. Results were tabulated in both TSV and XLSX formats for accessibility. In instances where raw sequence data were available, but not pre-annotated, read-level data extraction was conducted using the Regulatory Genomics Toolbox (RGT)⁵⁰.

Author contributions

All authors contributed to the study conception and design. A.T.B.S. and J.C.O.C.F. was responsible for the initial drafting of the manuscript. A.T.B.S., D.L.S.S., F.M. and K.T.C.C. participated in the material preparation, data collection, and analysis. All authors critically reviewed and provided feedback on previous versions of the manuscript. G.A.L., R.N.C., K.M.G.L., N.L.S., E.A.D. made significant contributions to revising and finalizing the manuscript. All authors read and approved the final manuscript.

Data availability

The datasets generated and/or analysed during the current study are available in the European Nucleotide Archive (ENA) repository, https://www.ebi.ac.uk/ena/browser/view/ERP165468.

Declarations

Competing interests

The authors declare no competing interests.